Serial filtration to generate small cholesterol-containing liposomes

ABSTRACT

The present invention provides methods and systems for producing liposomes by filtration. The methods include passing a heated lipid suspension through a filter assembly comprising two or more filters connected in series, wherein an orifice is disposed between adjacent filters. The methods and systems can produce liposomes having an average diameter that is less than half the diameter of the filter pores. Further, the methods and systems can produce liposomes with &lt;100 nm average diameter, even when the liposomes comprise at least  30 % sterol.

BACKGROUND OF THE INVENTION

The present application claims priority to U.S. Provisional Pat. Appl.No. 62/355,552, filed on Jun. 28, 2016, which application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Nanoparticles are of great interest for future developments in drugdelivery in human subjects. The ideal size range of a nanoparticle tomaximize bioavailability is between about 20 and about 100 nanometers(nm) in diameter. This size range is above renal clearance but belowhepatic fenestration and splenic sinusoidal clearance. Liposomes are ofparticular interest for delivery of therapeutic agents due to theirbiocompatibility and their ability to deliver polar and non-polartherapeutic agents. Liposomes suitable for drug delivery often requirecholesterol or a cholesterol derivative to hold together the lipidbilayer, in order to prevent lipid ejection and protein penetration thatlead to subsequent opsonization and macrophage clearance from thebloodstream. At least around 30 mole percent of cholesterol is typicallyrequired for a serum-stable formulation. However, cholesterol-containingliposomes having a diameter of less than 100 nm have proven difficult toprepare.

Existing methods for producing liposomes include extrusion, probesonication, and bath sonication. Extrusion relies on multiple passes ofa lipid suspension from one syringe to another, through a membrane witha given pore size. This method is limited to the production of smallbatches of liposomes due to flow rate constraints. Further, themembranes are very prone to clogging, resulting in significant materialloss and low yield. Also, extrusion is not effective for producing <100nm liposomes from a lipid suspension comprising significant amounts ofcholesterol, such as about 30 mole percent. Probe sonication is not usedcommercially because probe materials such as titanium can slough off ofthe probe and contaminate the lipid suspension. Bath sonication haslimited power and ability to consistently generate liposomes below 100nm unless coupled with a second processes step such as syringefiltration or extrusion. Thus, new methods are needed for preparation ofuniformly small liposomes containing cholesterol.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, provided is a method of producing liposomes. Themethod comprises the steps of providing a lipid suspension comprisingone or more component lipids; heating the lipid suspension to atemperature which is above the phase transition temperature of thecomponent lipids; and passing the heated lipid suspension through afilter assembly. The filter assembly may comprise two or more filtersconnected in series with an orifice disposed between adjacent filters.This method can produce liposomes that have a diameter that issignificantly lower than the pore size of the filters. For example, inan embodiment, a ratio of the average filter pore size to the averageliposome diameter is 1.6 or greater. Liposomes produced by this methodcan have an average diameter of less than about 100 nm, even when theliposomes comprise cholesterol in amount of at least 30 mole percent.Further, the liposomes can have polyethylene glycol (PEG) brushdensities of >100,000 PEG chains per liposome having an average diameterof 100 nm.

In another aspect, provided are liposomes that comprise a sterol inamount of at least 30 mole percent, wherein the average diameter of theliposomes is less than 100 nm. The liposomes may have polyethyleneglycol brush densities of >100,000 polyethylene glycol chains perliposome having an average diameter of 100 nm. A population of suchliposomes can have a polydispersity index of 0.20 or less.

In another aspect, provided are systems for producing liposomes from alipid suspension. In some embodiments, a system for producing liposomesfrom a lipid suspension comprises a filter assembly. In someembodiments, the filter assembly comprises two or more filters disposedin series with an orifice disposed between each pair of adjacentfilters. In some embodiments, the system further comprises a componentto move the lipid suspension through the filters. The system can have aflow rate of 30 mL/minute or greater. The system does not have to beheated to produce liposomes. Further, the system can be sized for astatic volume ranging from small microliter-scale volumes, or less, tothousands of liters, or more. A benefit of serial filtration is thatfilters with larger pores may be used to generate relatively smallerliposomes, thus preventing filter clogging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a system of the invention.

FIG. 2 depicts another system of the invention.

FIG. 3 depicts average particle size and polydispersity of liposomesfiltered through 1, 2, or 4 filters (shown as curves labeled 1, 2, and4, respectively) in series as determined by dynamic light scattering(DLS).

FIG. 4 depicts the reproducible preparation of populations of ˜50 nm,˜100 nm, and ˜200 nm diameter liposomes, as determined by DLS.

FIG. 5A shows that changing the PEG loading of liposomes containingDPSE-PEG5000 by varying the mole percent of the PEG lipid does notchange the particle size of liposomes, as determined by DLS. DLS data isshown for liposomes containing 1.5 mol %, 2.5 mol %, and 5 mol %DSPE-PEG5000.

FIG. 5B shows the linear control of PEG loading (at 1.5 mol %, 2.5 mol%, and 5 mol %), as determined by NMR.

FIG. 6 shows that changing the PEG length (2000 g/mol vs. 5000 g/mol)does not change the particle size of liposomes, as determined by DLS.

FIG. 7 shows that average liposome particle size decreases withincreasing number of filters in series, and with increasing number ofpasses through those filters, as determined by DLS. The x-axis valuesindicate the number of passes and the number of filters in series (e.g.,1×1), respectively.

FIG. 8 shows that average liposome particle size decreases withincreasing number of passes through two filters in series, as determinedby DLS.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are novel methods and systems for consistently producinghighly mono-disperse, small-diameter (<100 nm diameter) liposomes havingabout 20-40 mole percent sterol. Also provided are populations ofliposomes generated using the methods and systems disclosed herein.Surprisingly, filtering a cholesterol-containing lipid suspensionthrough filters connected in series with an orifice disposed betweenadjacent filters provides liposomes that are smaller in diameter andmore monodisperse than liposomes prepared with the same number offilters not connected in series. Further, the average diameter of theliposomes produced via serial filtration can be significantly smaller indiameter than the size of the pores of the filters, such as producing50-nm liposomes by serial filtration through filters having 100 nmpores.

As used herein, the term “liposome” encompasses any compartment enclosedby a lipid bilayer. The term liposome includes unilamellar vesicleswhich are comprised of a single lipid bilayer and generally have adiameter in the range of about 20 nm to 10 μm. “Small unilamellarvesicles,” or SUVs typically range from about 20 nm to about 200 nm insize. Liposomes can also be multilamellar, which generally have adiameter in the range of 1 to 10 μm.

As used herein, the terms “liposome size” and “average particle size”refer to the outer diameter of a liposome. Average particle size can bedetermined by a number of techniques including dynamic light scattering(DLS), quasi-elastic light scattering (QELS), and electron microscopy.

As used herein, the term “polydispersity index” refers to the sizedistribution of a population of liposomes. Polydispersity index can bedetermined by a number of techniques including dynamic light scattering(DLS), quasi-elastic light scattering (QELS), and electron microscopy.Polydispersity index (PDI) is usually calculated as:

${PDI} = \left( \frac{\sigma}{d} \right)^{2}$

i.e., the square of (standard deviation/mean diameter).

As used herein, the term “lipid” refers to lipid molecules that caninclude fats, waxes, steroids, cholesterol, fat-soluble vitamins,monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids,cationic or anionic lipids, derivatized lipids, and the like. Exemplaryliposomes as described in detail below. Lipids can form micelles,monolayers, and bilayer membranes. The lipids can self-assemble intoliposomes.

As used herein, the term “amphiphilic lipid” refers to lipid moleculesthat contain a polar region and a non-polar region. In general, lipidshave a polar head group and a non-polar tail group.

As used herein, the term “phosphatidylcholine” refers to adiacylglyceride phospholipid having a choline headgroup (i.e., a1,2-diacyl-sn-glycero-3-phosphocholine). The acyl groups in aphosphatidylcholine lipid are generally derived from fatty acids havingfrom 6 to 24 carbon atoms. Phosphatidylcholine lipids can includesynthetic and naturally-derived 1,2-diacyl-sn-glycero-3-phosphocholines.

As used herein, the term “sterol” refers to a steroid containing atleast one hydroxyl group. A steroid is characterized by the presence ofa fused, tetracyclic gonane ring system. Sterols include, but are notlimited to, cholesterol (i.e.,2,15-dimethyl-14-(1,5-dimethylhexyl)-tetracyclo[8.7.0.0^(2,7).0^(11,15)]heptacos-7-en-5-ol; Chemical Abstracts Services Registry No.57-88-5).

As used herein, the term “PEG-lipid” refers to a poly(ethylene glycol)polymer covalently bonded to a hydrophobic or amphiphilic lipid moiety.The lipid moiety can include fats, waxes, steroids, fat-solublevitamins, monoglycerides, diglycerides, phospholipids, andsphingolipids. For example, the PEG-lipid may be adiacyl-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)] or anN-acyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}. Themolecular weight of the PEG in the PEG-lipid is generally from about 500to about 5000 Daltons (Da; g/mol). The PEG in the PEG-lipid can have alinear or branched structure.

As used herein, the term “PEG brush density” refers to the number of PEGchains extending from the surface of a liposome, expresses as a functionof liposome size. For example, a population of 100-nm liposomes havingon average 10,000 PEG chains per liposome has a PEG brush density of10,000 PEG chains per 100 nm liposome.

As used herein, the terms “molar percentage” and “mol %” refer to thenumber of a moles of a given lipid component of a liposome divided bythe total number of moles of all lipid components. Unless explicitlystated, the amounts of active agents, diluents, or other components arenot included when calculating the mol % for a lipid component of aliposome.

As used herein, the term “composition” refers to a product comprisingthe specified components in the specified amounts, as well as anyproduct which results, directly or indirectly, from combination of thespecified components in the specified amounts. Lipid compositions of thepresent invention generally contain a sterol and are pharmaceuticallyacceptable. By “pharmaceutically acceptable,” it is meant that thecarrier, diluent, or excipient must be compatible with the othercomponents of the formulation and non-deleterious to the recipientthereof.

As used herein, the term “porous filter” refers to a polymeric orinorganic membrane containing pores with a defined diameter (e.g.,50-500 nm). Porous filters can be made of polymers including, but notlimited to, polycarbonates and polyesters, as well as inorganicsubstrates including, but not limited to, porous alumina.

As used herein, the term “filters connected in series” refers to two ormore filter membranes that are placed near each other and separated bysome distance. Therefore, a fluid flowing through the filters would flowthrough a first filter and then flow through additional filters insequence.

As used herein, the term “orifice” refers to a physical feature such asan orifice, hole, opening, aperture, slit, or slot disposed between twofilter membranes. As used herein, the orifice generally has across-sectional area that is smaller than the average cross-sectionalarea of the adjacent filter membranes. An orifice can be contained in asystem component such as a filter housing fully enclosing a filtermembrane, or a panel (e.g., a disc or other shape) placed between filtermembranes but not enclosing the filter membranes.

As used herein, the term “static volume” refers to the volume of fluidrequired to fill an assembly or system when the fluid is not movingthrough the system.

As used herein, the terms “delivery” and “delivering” refer toconveyance of a therapeutic agent to a subject using the methods of theinvention. Delivery may be localized to a particular location in asubject, such as a tissue, an organ, or cells of a particular type.

As used herein, the term “therapeutic agent” refers to a compound ormolecule that, when present in an effective amount, produces a desiredtherapeutic effect in a subject in need thereof. The present inventioncontemplates a broad range of therapeutic agents and their use inconjunction with the liposome compositions.

As used herein, the term “subject” refers to any mammal, in particular ahuman, at any stage of life.

As used herein, the term “consists essentially of” refers to acomposition having the stated components, in addition to minorcomponents (e.g., unavoidable impurities) that do not materially affectthe properties of the composition (e.g., the average size ormonodispersity of a population of liposomes).

As used herein, the term “about” indicates a close range around anumerical value when used to modify that specific value. If “X” were thevalue, for example, “about X” would indicate a value from 0.9X to 1.1X,e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or avalue from 0.99X to 1.01X. Any reference to “about X” specificallyindicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X,1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.

I. METHODS

In a first aspect, provided is a method of producing liposomes. Themethod may comprise the steps of providing a lipid suspension comprisingone or more component lipids; heating the lipid suspension to atemperature which is above the phase transition temperature of thecomponent lipids; and passing the heated lipid suspension through afilter assembly. The filter assembly may comprise two or more filtersconnected in series with an orifice disposed between adjacent filters.The method allows for the reproducible production of liposomes ofcontrolled diameter with defined polydispersity.

Each filter may comprise a porous membrane containing pores with aspecified diameter or pore size. The filter pore diameter or pore sizemay be any size suitable for producing nanoparticle liposomes (e.g.,50-500 nm). For example, the filter pore diameter or pore size can befrom 50-100 nm, from 100-200 nm, or from 200-500 nm. In some instances,the filter pore diameter or pore size can be around 100 nm.

The porous filter membranes can be made of any inert material. Incertain embodiments, the filter(s) may comprise polymers including, butnot limited to, polycarbonates and polyesters. For example, the porousfilters may comprise GHP (hydrophilic polypropylene), PES (polyethersulfone), Nylon, or PVDF (polyvinylidene fluoride). Additionally and/oralternatively, the filter(s) may comprise inorganic substratesincluding, but not limited to, porous alumina or glass fiber. In someembodiments, the filter membranes are enclosed in a housing.

The filter assembly can contain two, three, four, five filters, andoptionally additional filters. In some embodiments, the filters aresyringe filters. In some cases, the filters are syringe-tip filters suchas an Acrodisc syringe filter with a Supor membrane (e.g., 13 mmdiameter, 25 mm diameter, or 32 mm diameter) sold by Pall Corporation,Port Washington, N.Y.

The filters may be connected in series, with two or more filtermembranes facing each other and separated by a distance typicallyranging from a few microns to several millimeters or more. In this way,a fluid flowing through the filters would flow through one filter, flowthrough an orifice disposed between the filters, and then flow throughthe additional filter(s). The distance between the filters can be anydistance greater than about 1000 nm. In certain embodiments, themembranes may be separated by 1 mm, 1 cm, 100 cm, or more. In general,the filter membranes are not stacked directly one on top of the otherand do not touch each other.

In various embodiments, at least one orifice is disposed betweenadjacent filters. The orifice is disposed between two adjacent filtermembranes so as to constrict the flow of fluid (e.g., the lipidsuspension). In various embodiments, the orifice is a hole, opening,aperture, slit, slot, or any similar means for constricting or narrowingthe diameter of the fluid stream as the fluid flows from an upstreamfilter to a downstream filter. Therefore, the orifice has a diameter (orcross-sectional area, if not round) that is smaller than the averagediameter (or cross-sectional area) of the adjacent filter membranes.

Not intending to be bound by theory, it is believed that theconstriction of the fluid stream between two adjacent filters introducesshear forces that promote formation of smaller liposomes than would havebeen formed by passing the same lipid suspension twice through a singlefilter. The resulting liposomes are also smaller liposomes than thoseproduced by passing the same lipid suspension through two filters thatare not connected, or that are connected without an orifice disposedbetween. Similarly, liposomes produced by filtration through 3 filtersin series will be smaller than liposomes produced by filtration through3 filters that are not in series, liposomes produced by filtrationthrough 4 filters in series will be smaller than liposomes produced byfiltration through 4 filters that are not in series, liposomes producedby filtration through 5 filters in series will be smaller than liposomesproduced by filtration through 5 filters that are not in series, and soon. In some examples, the diameter or cross-sectional area of eachorifice disposed between two adjacent filters is less than or equal toabout 1%, 5%, 20%, 50%, 70, or 80% of the average diameter orcross-sectional area of the filters adjacent to the orifice. In someembodiments, the diameter or cross-sectional area of each orificedisposed between two adjacent filters is less than or equal to 70% ofthe average diameter or cross-sectional area of the filters adjacent tothe orifice.

As described in detail below, for example, one pass of a lipidsuspension through 4 filters with orifices disposed between the filtersyielded liposomes having an average diameter of 60.49 nm and apolydispersity of 0.084, whereas passing the lipid suspension throughfour membrane filters stacked with no intervening orifices yieldedliposomes with an average diameter of 88.25 nm and a polydispersity of0.150. These results occurred even though each lipid suspension waspassed through the same number of filters.

Previously known systems for liposome preparation often require theapplication of significant pressure to force lipid suspensions throughfilters. In contrast, the filter assembly disclosed herein does notrequire significant pressurization to force the lipid suspension throughthe filter membrane. Although a small amount of pressure, e.g., around100 pounds per square inch (psi), can inherently build as the fluidflows or is pushed through the filter assembly, the method generallydoes not require applying higher pressures, e.g., 300 psi, 500 psi, 1000psi, or greater, as required by many previously-known methods. In someembodiments, the pressure in the filter assembly is less than 300 psi,less than 200 psi, or less than 100 psi.

In some embodiments, a lipid suspension is prepared by adding thedesired lipids in the desired molar ratios to an aqueous solvent, andthen providing mechanical energy sufficient to suspend the lipids in theaqueous solvent. The mechanical energy may be supplied by shaking,stirring, sonicating, or other similar methods.

The lipid suspension can contain any suitable lipid, including neutrallipids, cationic lipids, anionic lipids, and/or zwitterionic lipids.Suitable lipids can include fats, waxes, steroids, sterols, cholesterol,fat-soluble vitamins, monoglycerides, diglycerides, phospholipids,sphingolipids, glycolipids, cationic or anionic lipids, derivatizedlipids, and the like.

In some embodiments, the lipid suspension comprises an amphiphiliclipid, a sterol, and a (polyethylene glycol)-lipid. In some examples,the amphiphilic lipid is a phosphatidylcholine lipid. Suitablephosphatidylcholine lipids (PCs) include saturated PCs and unsaturatedPCs. Examples of saturated PCs include1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphatidylcholine; DMPC),1,2-distearoyl-sn-glycero-3-phosphocholine(distearoylphosphatidylcholine; DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine(dipalmitoylphosphatidylcholine; DPPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), and1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC).

Examples of unsaturated PCs include, but are not limited to,1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine,1,2-dipamiltoleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dielaidoyl-sn-glycero-3-phosphocholine,1,2-dipetroselenoyl-sn-glycero-3-phosphocholine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(palmitoyloleoylphosphatidylcholine; POPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC),1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC). Lipid extracts,such as egg PC, heart extract, brain extract, liver extract, soy PC, andhydrogenated soy PC (HSPC) can also be used in the methods of theinvention.

In some examples, the amphiphilic lipid is selected from the groupconsisting of hydrogenated soy phosphatidylcholine,1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphatidylcholine; DMPC),1,2-distearoyl-sn-glycero-3-phosphocholine(distearoylphosphatidylcholine; DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine(dipalmitoylphosphatidylcholine; DPPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC),1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine,1,2-dipamiltoleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dielaidoyl-sn-glycero-3-phosphocholine,1,2-dipetroselenoyl-sn-glycero-3-phosphocholine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(palmitoyloleoylphosphatidylcholine; POPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC),1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC).

The lipid suspensions provided herein will, in some embodiments, consistessentially of PC/cholesterol/PEG-lipid mixtures. In some embodiments,the liposome suspensions will consist essentially of aphosphatidylcholine lipid or mixture of phosphatidylcholine lipids, witha sterol, and a PEG-lipid. In some embodiments, when a single type ofphosphatidylcholine lipid is used, it is selected from DOPC, DSPC, HSPC,DPPC, POPC and SOPC.

In some embodiments, the phosphatidylcholine lipid is selected from thegroup consisting of DPPC, DSPC, HSPC, and mixtures thereof. Any suitableamount of phosphatidylcholine or phosphatidylcholine mixture can be usedin the lipid suspension. For example, the amount of phosphatidylcholineor phosphatidylcholine mixture in the lipid suspension can range fromabout 40 mol % to about 43 mol %, or from about 43 mol % to about 46 mol%, or from about 46 mol % to about 49 mol %, or from about 49 mol % toabout 52 mol %, or from about 52 mol % to about 55 mol %, or from about55 mol % to about 58 mol %, or from about 58 mol % to about 61 mol %, orfrom about 61 mol % to about 64 mol %, or from about 64 mol % to about67 mol %, or from about 67 mol % to about 70 mol %. The amount ofphosphatidylcholine or phosphatidylcholine mixture in the lipidsuspension can range from about 40 mol % to about 70 mol %, or fromabout 42 mol % to about 68 mol %, or from about 44 mol % to about 66 mol%, or from about 46 mol % to about 64 mol %, or from about 48 mol % toabout 62 mol %, or from about 50 mol % to about 60 mol %, or from about52 mol % to about 58 mol %, or from about 54 mol % to about 56 mol %. Insome embodiments, the liposomes contain 50-65 mol % of aphosphatidylcholine lipid or mixture of phosphatidylcholine lipids, or45-70 mol % of a phosphatidylcholine lipid or mixture ofphosphatidylcholine lipids. The liposomes can contain, for example, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mol %phosphatidylcholine. In some embodiments, the lipid suspensions containabout 56 mol % phosphatidylcholine.

Other suitable phospholipids, generally used in low amounts or inamounts less than the phosphatidylcholine lipids, include phosphatidicacids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols(PGs), phosphatidylserine (PSs), and phosphatidylinositol (PIs).Examples of phospholipids include, but are not limited to,1,2-distearoyl-sn-glycero-3-phosphate (DSPA),dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylserine(DMPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine(DOPS), dipalmitoylphosphatidylserine (DPPS),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dipalmitoylphosphatidylethanolamine (DPPE),dimyristoylphosphoethanolamine (DMPE),distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),dielaidoylphosphoethanolamine (transDOPE), and cardiolipin.

In some embodiments, phospholipids can include reactive functionalgroups for further derivatization. Examples of such reactive lipidsinclude, but are not limited to,dioleoylphospha-tidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(DOPE-mal) and dipalmitoylphosphatidylethanolamine-N-succinyl(succinyl-PE).

In general, the lipid suspensions contain at least one sterol. In somecases, the sterol is cholesterol or a cholesterol derivative, such as2,15-dimethyl-14-(1,5-dimethylhexyl)tetracyclo[8.7.0.0^(2,7).0^(11,15)]heptacos-7-en-5-ol) or cholesteryl pelargonate. Other sterols,including stigmasterol, campesterol, zymostenol, sitosterol, andpregnenolone, can also be used in the lipid suspension. Any suitableamount of sterol can be used in the lipid suspension. For example, theamount of the sterol or sterol mixture in the lipid suspension can rangefrom about 20 mol % to about 24 mol %, or from about 24 mol % to about28 mol %, or from about 28 mol % to about 32 mol %, or from about 32 mol% to about 36 mol %, or from about 36 mol % to about 40 mol %, or fromabout 40 mol % to about 44 mol %, or from about 44 mol % to about 48 mol%, or from about 48 mol % to about 50 mol %. The amount of the sterol orsterol mixture in the lipid suspension can range from about 20 mol % toabout 50 mol %, or from about 23 mol % to about 47 mol %, or from about26 mol % to about 44 mol %, or from about 29 mol % to about 41 mol %, orfrom about 32 mol % to about 38 mol %, or from about 35 mol % to about35 mol %, or from about 38 mol % to about 32 mol %, or from about 41 mol% to about 29 mol %, or from about 44 mol % to about 26 mol %, or fromabout 47 mol % to about 23 mol %, or from about 50 mol % to about 20 mol%. In some embodiments, the liposomes can contain about 20-50 mol %sterol, or about 25-35 mol % sterol. The liposomes can contain, forexample, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45 mol % sterol. In some embodiments, the lipidsuspensions contain 30-40 mol % cholesterol. In some embodiments, theliposomes contain 20-30 mol % cholesterol. In some embodiments, thelipid suspensions contain 30 mol % cholesterol. In some embodiments, thelipid suspensions contain 39 mol % cholesterol. Lipid suspensions of thepresent invention can contain other steroids, characterized by thepresence of a fused, tetracyclic gonane ring system. Examples ofsteroids include, but are not limited to, cholic acid, progesterone,cortisone, aldosterone, testosterone, dehydroepiandrosterone, andestradiol. Synthetic steroids and derivatives thereof are alsocontemplated for use in the present invention.

In some embodiments, the lipid suspensions also contain a (polyethyleneglycol)-lipid. The presence of PEG on the surface of a liposome has beenshown to extend blood-circulation time while reducing mononuclearphagocyte system uptake, creating so-called “stealth” liposomes asdescribed in U.S. Pat. Nos. 5,013,556 and 5,827,533, each of which isincorporated herein by reference in its entirety. In some embodiments,the (polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine. In some embodiments, the liposomesproduced by the method have an average diameter of 100 nm andpolyethylene glycol brush densities of greater than 25,000 polyethyleneglycol chains per liposome, or greater than 50,000 polyethylene glycolchains per liposome, or greater than 100,000 polyethylene glycol chainsper liposome, or greater than 200,000 polyethylene glycol chains perliposome, or greater than 250,000 polyethylene glycol chains perliposome. In some embodiments, the liposomes have an average diameter of50 nm and polyethylene glycol brush densities of greater than 25,000polyethylene glycol chains per liposome, or greater than 50,000polyethylene glycol chains per liposome, or greater than >100,000polyethylene glycol chains per liposome, or greater than 250,000polyethylene glycol chains per liposome. In some embodiments, theliposomes comprising a sterol in amount of at least 30 mole percent havepolyethylene glycol brush densities of >100,000 polyethylene glycolchains per liposome having an average diameter of 100 nm.

The lipid suspensions may include any suitable poly(ethyleneglycol)-lipid derivative (PEG-lipid). In some embodiments, the PEG-lipidis a diacyl-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)].The molecular weight of the poly(ethylene glycol) in the PEG-lipid isgenerally in the range of from about 500 Daltons (Da) to about 5000 Da.The poly(ethylene glycol) can have a molecular weight of, for example,about 750 Da, about 1000 Da, about 2500 Da, or about 5000 Da, or about10,000 Da, or any molecular weight within this range. In someembodiments, the PEG-lipid is selected fromdistearoyl-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2500] (DSPE-PEG-2500) anddistearoyl-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG-5000). In some embodiments, the PEG-lipid isDSPE-PEG-2500. In other embodiments, the PEG-lipid is DSPE-PEG-5000.

Any suitable amount of PEG-lipid can be used in the lipid suspension.For example, the amount of the PEG-lipid in the lipid suspension canrange from about 1 mol % to about 2 mol %, or from about 2 mol % toabout 3 mol %, or from about 3 mol % to about 4 mol %, or from about 4mol % to about 5 mol %, or from about 5 mol % to about 6 mol %, or fromabout 6 mol % to about 7 mol %, or from about 7 mol % to about 8 mol %,or from about 8 mol % to about 9 mol %, or from about 9 mol % to about10 mol %. The amount of the PEG-lipid in the lipid suspension can rangefrom about 1 mol % to about 10 mol %, or from about 2 mol % to about 9mol %, or from about 3 mol % to about 8 mol %, or from about 4 mol % toabout 7 mol %. In some embodiments, the lipid suspensions contain 1-8mol % of the PEG-lipid. The liposomes can contain, for example, 1, 2, 3,4, 5, 6, 7, or 8 mol % PEG-lipid. In some embodiments, the liposomescontain 2-6 mol % PEG-lipid. In some embodiments, the liposomes contain3 mol % PEG-lipid. In some embodiments, the liposomes contain 5 mol %DSPE-PEG-2000.

In some examples, the lipid suspensions comprise a sterol in an amountof at least 30 mole percent and further comprise an amphiphilic lipidand a (polyethylene glycol)-lipid. In some embodiments, the sterol ischolesterol or a cholesterol derivative. In some examples, the(polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine. In some instances, the amphiphiliclipid is hydrogenated soy phosphatidylcholine, the sterol ischolesterol, and the (polyethylene glycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K.

In some embodiments, the lipid suspension contains from about 50 mol %to about 65 mol % HSPC, from about 35 mol % to about 45 mol %cholesterol, and from about 2 mol % to about 8 mol % DSPE-PEG5K. In someembodiments, the lipid suspension contains from about 50 mol % to about70 mol % DPPE, from about 30 mol % to about 40 mol % cholesterol, andfrom about 2 mol % to about 8 mol % DSPE-PEG5K.

In some embodiments, the lipid suspension contains from about 50 mol %to about 70 mol % DPPE, from about 30 mol % to about 40 mol %cholesterol, from about 0.1 mol % to about 0.5 mol % DPPE-Cy5.5, andfrom about 1.5 mol % to about 8 mol % DSPE-PEG5K. In some embodiments,the lipid suspension contains from about 50 mol % to about 65 mol %HSPC, from about 35 mol % to about 45 mol % cholesterol, from about 0.1mol % to about 0.5 mol % DPPE-Cy5.5, and from about 1.5 mol % to about 8mol % DSPE-PEG5K.

In some embodiments, the amphiphilic lipid is hydrogenated soyphosphatidylcholine, the sterol is cholesterol, and the (polyethyleneglycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K. In some embodiments, a 56:39:5 molar ratio ofHSPC:cholesterol:DSPE-PEG5K is used. In some embodiments, a 60:35:5molar ratio of DPPE:cholesterol:DSPE-PEG5K is used. In some embodiments,a 60:35:0.1:4.9 molar ratio of DPPE:cholesterol:DPPE-Cy5.5:DSPE-PEG5K isused. In some embodiments, a 60:35:0.1:4.9 molar ratio ofHSPC:cholesterol:DPPE-Cy5.5:DSPE-PEG5K is used.

Further lipids can be included in the lipid suspensions and liposomesincluding, but not limited to, quanternary amine-based cationic lipids(e.g., 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),Dimethyldioctadecylammonium bromide salt (DDAB), and the like),diacylglycerols (e.g., 1,2-dipalmitoyl-sn-glycerol, glycerol distearate,and the like); alkyl phosphates(1,2-dipalmitoyl-sn-glycero-3-phosphomethanol,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, and thelike); and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethylphosphate (DOCP).

The methods provided are particularly useful for producing liposomeshaving an average diameter of less than about 100 nm. The diameter ofthe liposomes can range, for example, from about 5 nm to about 10 nm, orfrom about 10 nm to about 20 nm, or from about 20 nm to about 30 nm, orfrom about 30 nm to about 40 nm, or from about 40 nm to about 50 nm, orfrom about 50 nm to about 60 nm, or from about 60 nm to about 70 nm, orfrom about 70 nm to about 80 nm, or from about 80 nm to about 90 nm, orfrom about 90 nm to about 95 nm. The diameter of the liposomes can rangefrom about 35 nm to about 40 nm, or from about 40 nm to about 45 nm, orfrom about 45 nm to about 50 nm, or from about 40 nm to about 60 nm, orfrom about 20 nm to about 80 nm, or from about 10 nm to about 90 nm. Thediameter of the liposomes can be about 40, 41, 42, 43, 44, 45, 46, 47,48, 49 50, 51, 52, 53, 54, or 55 nm. Liposomes of this size have provendifficult to produce via previously known methods, particularly when asterol such as cholesterol is incorporated in about 20-40 mol %, or inabout 30 mol %, or in greater than 30 mol %.

The methods provided are also particularly useful for producingliposomes having an average diameter that is substantially less than thepore diameter of the filter membranes. Thus, for instance, a 100 nm-porefilter membrane can be used to produce liposomes having a diameter of 50nm. In some embodiments, the ratio of the average filter pore size tothe average liposome diameter is 1.2 or greater, 1.4 or greater, 1.6 orgreater, 1.8 or greater, 2.0 or greater, or 3.0 or greater. In someembodiments, the ratio of the average filter pore size to the averageliposome diameter is from 1.2 to 3.0, or from 1.6 to 2.0. In someembodiments, the average pore size of the filters is about 100 nm andthe average diameter of the liposomes is about 80 nm, about 70 nm, about60 nm, about 50 nm, or about 45 nm.

The liposome populations produced via the methods described herein havelow polydispersities, generally having a polydispersity index that isless than 0.3, less than 0.2, less than 0.15, or less than 0.10, asmeasured by DLS.

The liposome populations produced via the methods described herein maybe unilamellar vesicles which are comprised of a single lipid bilayerand generally have a diameter in the range of about 20 to about 400 nm.Liposomes can also be multilamellar, which generally have a diameter inthe range of 1 to 10 μm. In some embodiments, liposomes can includemultilamellar vesicles (MLVs; from about 1 μm to about 10 μm in size),large unilamellar vesicles (LUVs; from a few hundred nanometers to about10 μm in size), and/or small unilamellar vesicles (SUVs; from about 20nm to about 200 nm in size). In some instances, the liposomes are smallunilamellar vesicles.

In some examples, the liposomes are produced when the lipid suspensionis passed through the filter assembly more than once. In someembodiments, the average diameter of the liposomes decreases with eachadditional pass through the filter assembly.

Yield can be used to express the amount of lipids from the lipidsuspension that are not lost during filtration. In some embodiments, thelipid suspension is converted to liposomes with a yield of at leastabout 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), atleast about 80% (w/w), or at least about 90% (w/w).

II. LIPOSOMES

A variety of liposomes can be prepared using the methods and systemsdescribed herein. In certain embodiments, liposomes that comprise asterol in amount of at least 30 mole percent, wherein the averagediameter of the liposomes is less than 100 nm, are provided.

In some embodiments, liposomes are prepared from a lipid suspension. Thelipid suspension is prepared by adding the desired lipids in the desiredmolar ratios to an aqueous solvent, and then dispersing the lipids inthe solvent. The lipid suspension can contain any suitable lipid,including cationic lipids, zwitterionic lipids, neutral lipids, oranionic lipids as described above. Suitable lipids can include fats,waxes, steroids, sterols, cholesterol, fat-soluble vitamins,monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids,cationic or anionic lipids, derivatized lipids, and the like.

In some embodiments, the liposomes comprise an amphiphilic lipid, asterol, and a (polyethylene glycol)-lipid. In some examples, theamphiphilic lipid is a phosphatidylcholine lipid. Suitablephosphatidylcholine lipids include saturated PCs and unsaturated PCs.Examples of saturated PCs include1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphatidylcholine; DMPC),1,2-distearoyl-sn-glycero-3-phosphocholine (distearoylphosphatidylcholine; DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine(dipalmitoylphosphatidylcholine; DPPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), and1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC).

Examples of unsaturated PCs include, but are not limited to,1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine,1,2-dipamiltoleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dielaidoyl-sn-glycero-3-phosphocholine,1,2-dipetroselenoyl-sn-glycero-3-phosphocholine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(palmitoyloleoylphosphatidylcholine; POPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC),1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC), and lipidextracts, such as egg PC, heart extract, brain extract, liver extract,soy PC, and hydrogenated soy PC (HSPC) are also useful in the presentinvention.

In some embodiments, the amphiphilic lipid is selected from the groupconsisting of hydrogenated soy phosphatidylcholine,1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphatidylcholine; DMPC),1,2-distearoyl-sn-glycero-3-phosphocholine(distearoylphosphatidylcholine; DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine(dipalmitoylphosphatidylcholine; DPPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC),1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine,1,2-dipamiltoleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dielaidoyl-sn-glycero-3-phosphocholine,1,2-dipetroselenoyl-sn-glycero-3-phosphocholine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(palmitoyloleoylphosphatidylcholine; POPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC),1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC). Lipid extracts,such as egg PC, heart extract, brain extract, liver extract, soy PC, andhydrogenated soy PC (HSPC).

The liposomes provided herein will, in some embodiments, consistessentially of PC/cholesterol/PEG-lipid mixtures. In some embodiments,the liposomes will consist essentially of a phosphatidylcholine lipid ormixture of phosphatidylcholine lipids, with a sterol, and a PEG-lipid.In some embodiments, when a single type of phosphatidylcholine lipid isused, it is selected from DOPC, DSPC, HSPC, DPPC, POPC and SOPC.

In some embodiments, the phosphatidylcholine lipid is selected from thegroup consisting of DPPC, DSPC, HSPC, and mixtures thereof. Theliposomes can contain any suitable amount of phosphatidylcholine orphosphatidylcholine mixture. For example, the amount ofphosphatidylcholine or phosphatidylcholine mixture in the liposomes canrange from about 40 mol % to about 43 mol %, or from about 43 mol % toabout 46 mol %, or from about 46 mol % to about 49 mol %, or from about49 mol % to about 52 mol %, or from about 52 mol % to about 55 mol %, orfrom about 55 mol % to about 58 mol %, or from about 58 mol % to about61 mol %, or from about 61 mol % to about 64 mol %, or from about 64 mol% to about 67 mol %, or from about 67 mol % to about 70 mol %. Theamount of phosphatidylcholine or phosphatidylcholine mixture in theliposomes can range from about 40 mol % to about 70 mol %, or from about42 mol % to about 68 mol %, or from about 44 mol % to about 66 mol %, orfrom about 46 mol % to about 64 mol %, or from about 48 mol % to about62 mol %, or from about 50 mol % to about 60 mol %, or from about 52 mol% to about 58 mol %, or from about 54 mol % to about 56 mol %. In someembodiments, the liposomes contain 50-65 mol % of a phosphatidylcholinelipid or mixture of phosphatidylcholine lipids or 45-70 mol % of aphosphatidylcholine lipid or mixture of phosphatidylcholine lipids. Theliposomes can contain, for example, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64 or 65 mol % phosphatidylcholine. In someembodiments, the liposomes contain about 56 mol % phosphatidylcholine.

Other suitable phospholipids, generally used in low amounts or inamounts less than the phosphatidylcholine lipids, include phosphatidicacids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols(PGs), phosphatidylserine (PSs), and phosphatidylinositol (PIs).Examples of phospholipids include, but are not limited to,1,2-distearoyl-sn-glycero-3-phosphate (DSPA),dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylserine(DMPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine(DOPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dipalmitoylphosphatidylethanolamine (DPPE),dimyristoylphosphoethanolamine (DMPE),distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),dielaidoylphosphoethanolamine (transDOPE), and cardiolipin.

In some embodiments, phospholipids can include reactive functionalgroups for further derivatization. Examples of such reactive lipidsinclude, but are not limited to,dioleoylphospha-tidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(DOPE-mal) and dipalmitoylphosphatidylethanolamine-N-succinyl(succinyl-PE).

In general, the liposomes contain at least one sterol. In some cases,the sterol is cholesterol or a cholesterol derivative, such as2,15-dimethyl-14-(1,5-dimethylhexyl)tetracyclo[8.7.0.0^(2,7).0^(11,15)]heptacos-7-en-5-ol) or cholesteryl pelargonate. Other sterols,including stigmasterol, campesterol, zymostenol, sitosterol, andpregnenolone, can also be included in the liposomes. The liposomes cancontain any suitable amount of sterol. For example, the amount of thesterol or sterol mixture in the liposomes can range from about 20 mol %to about 24 mol %, or from about 24 mol % to about 28 mol %, or fromabout 28 mol % to about 32 mol %, or from about 32 mol % to about 36 mol%, or from about 36 mol % to about 40 mol %, or from about 40 mol % toabout 44 mol %, or from about 44 mol % to about 48 mol %, or from about48 mol % to about 50 mol %. The amount of the sterol or sterol mixturein the liposomes can range from about 20 mol % to about 50 mol %, orfrom about 23 mol % to about 47 mol %, or from about 26 mol % to about44 mol %, or from about 29 mol % to about 41 mol %, or from about 32 mol% to about 38 mol %, or from about 35 mol % to about 35 mol %, or fromabout 38 mol % to about 32 mol %, or from about 41 mol % to about 29 mol%, or from about 44 mol % to about 26 mol %, or from about 47 mol % toabout 23 mol %, or from about 50 mol % to about 20 mol %. In someembodiments, the liposomes can contain about 20-50 mol % sterol, orabout 25-35 mol % sterol. The liposomes can contain, for example, 24,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, or 45 mol % sterol. In some embodiments, the liposomes contain 30-40mol % cholesterol. In some embodiments, the liposomes contain 20-30 mol% cholesterol. In some embodiments, the liposomes contain 30 mol %cholesterol. In some embodiments, the liposomes contain 39 mol %cholesterol. Liposomes of the present invention can contain othersteroids, characterized by the presence of a fused, tetracyclic gonanering system. Examples of steroids include, but are not limited to,cholic acid, progesterone, cortisone, aldosterone, testosterone,dehydroepiandrosterone, and estradiol. Synthetic steroids andderivatives thereof are also contemplated for use in the presentinvention.

In some embodiments, the liposomes also contain a (polyethyleneglycol)-lipid. The presence of PEG on the surface of a liposome has beenshown to extend blood-circulation time while reducing mononuclearphagocyte system uptake, creating so-called “stealth” liposomes asdescribed in U.S. Pat. Nos. 5,013,556 and 5,827,533, each of which ishereby incorporated by reference in its entirety. In some embodiments,the (polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine.

The liposomes may include any suitable poly(ethylene glycol)-lipidderivative (PEG-lipid). In some embodiments, the PEG-lipid is adiacyl-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]. Themolecular weight of the poly(ethylene glycol) in the PEG-lipid isgenerally in the range of from about 500 Daltons (Da) to about 5000 Da.The poly(ethylene glycol) can have a molecular weight of, for example,about 750 Da, about 1000 Da, about 2500 Da, or about 5000 Da, or about10,000 Da, or any molecular weight within this range. In someembodiments, the PEG-lipid is selected fromdistearoyl-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2500] (DSPE-PEG-2500) anddistearoyl-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG-5000). In some embodiments, the PEG-lipid isDSPE-PEG-2500. In other embodiments, the PEG-lipid is DSPE-PEG-5000.

The liposomes may contain any suitable amount of PEG-lipid. For example,the amount of the PEG-lipid in the liposomes can range from about 1 mol% to about 2 mol %, or from about 2 mol % to about 3 mol %, or fromabout 3 mol % to about 4 mol %, or from about 4 mol % to about 5 mol %,or from about 5 mol % to about 6 mol %, or from about 6 mol % to about 7mol %, or from about 7 mol % to about 8 mol %, or from about 8 mol % toabout 9 mol %, or from about 9 mol % to about 10 mol %. The amount ofthe PEG-lipid in the liposomes can range from about 1 mol % to about 10mol %, or from about 2 mol % to about 9 mol %, or from about 3 mol % toabout 8 mol %, or from about 4 mol % to about 7 mol %. In someembodiments, the liposomes contain 1-8 mol % of the PEG-lipid. Theliposomes can contain, for example, 1, 2, 3, 4, 5, 6, 7, or 8 mol %PEG-lipid. In some embodiments, the liposomes contain 2-6 mol %PEG-lipid. In some embodiments, the liposomes contain 3 mol % PEG-lipid.In some embodiments, the liposomes contain 5 mol % DSPE-PEG-2000.

In some embodiments, the amphiphilic lipid is hydrogenated soyphosphatidylcholine, the sterol is cholesterol, and the (polyethyleneglycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K. In some embodiments, a 56:39:5 molar ratio ofHSPC:cholesterol:DSPE-PEG5K is used. In other embodiments, a 60:35:5molar ratio of DPPE:cholesterol:DSPE-PEG5K is used. In addition, a60:35:0.1:4.9 molar ratio of DPPE:cholesterol:DPPE-Cy5.5:DSPE-PEG5K isused. In some embodiments, a 60:35:0.1:4.9 molar ratio ofHSPC:cholesterol:DPPE-Cy5.5:DSPE-PEG5K is used.

In some embodiments, the liposomes contain from about 50 mol % to about65 mol % HSPC, from about 35 mol % to about 45 mol % cholesterol, andfrom about 2 mol % to about 8 mol % DSPE-PEG5K. In some embodiments, theliposomes contain from about 50 mol % to about 70 mol % DPPE, from about30 mol % to about 40 mol % cholesterol, and from about 2 mol % to about8 mol % DSPE-PEG5K.

In some embodiments, the liposomes contain from about 50 mol % to about70 mol % DPPE, from about 30 mol % to about 40 mol % cholesterol, fromabout 0.1 mol % to about 0.5 mol % DPPE-Cy5.5, and from about 1.5 mol %to about 8 mol % DSPE-PEG5K. In some embodiments, the liposomes containfrom about 50 mol % to about 65 mol % HSPC, from about 35 mol % to about45 mol % cholesterol, from about 0.1 mol % to about 0.5 mol %DPPE-Cy5.5, and from about 1.5 mol % to about 8 mol % DSPE-PEG5K.

In some examples, the liposomes comprising a sterol in an amount of atleast 30 mole percent further comprise an amphiphilic lipid and a(polyethylene glycol)-lipid. In some embodiments, the sterol ischolesterol or a cholesterol derivative. In some examples, the(polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine. In some instances, the amphiphiliclipid is hydrogenated soy phosphatidylcholine, the sterol ischolesterol, and the (polyethylene glycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K.

In some embodiments, the liposomes have an average diameter of 100 nmand polyethylene glycol brush densities of >25,000 polyethylene glycolchains per liposome, >50,000 polyethylene glycol chains perliposome, >100,000 polyethylene glycol chains per liposome , >200,000polyethylene glycol chains per liposome, or >250,000 polyethylene glycolchains per liposome.

In some embodiments, the liposomes have an average diameter of 50 nm andpolyethylene glycol brush densities of >25 polyethylene glycol chainsper liposome, >50 polyethylene glycol chains per liposome, >100polyethylene glycol chains per liposome, or >250 polyethylene glycolchains per liposome. In some embodiments, the liposomes comprising asterol in amount of at least 30 mole percent have polyethylene glycolbrush densities of >100,000 polyethylene glycol chains per liposomehaving an average diameter of 100 nm.

The liposomes may be unilamellar vesicles which are comprised of asingle lipid bilayer and generally have a diameter in the range of about20 to about 400 nm. Liposomes can also be multilamellar, which generallyhave a diameter in the range of 1 to 10 μm. In some embodiments,liposomes can include multilamellar vesicles (MLVs; from about 1 μm toabout 10 μm in size), large unilamellar vesicles (LUVs; from a fewhundred nanometers to about 10 μm in size), and small unilamellarvesicles (SUVs; from about 20 nm to about 200 nm in size). In someinstances, the liposomes are small unilamellar vesicles.

In some embodiments, small liposomes (e.g., diameter of 100 nm or less)contain a 20-40 mol % sterol or sterol derivative. In some instances,liposomes comprising cholesterol in amount of at least 30 mole percenthave an average diameter of less than about 100 nm. In some cases,liposomes comprising cholesterol in amount of at least 30 mole percenthave an average diameter of less than about 75 nm. In other cases,liposomes comprising cholesterol in amount of at least 30 mole percenthave an average diameter of less than about 55 nm. In some instances,liposomes comprising cholesterol in amount of at least 30 mole percenthave an average diameter of less than about 50 nm.

In some embodiments, liposomes comprising cholesterol in amount of atleast 30 mole percent have a diameter that is the diameter determined bythe thermodynamic lower limit of the lipid composition. One of skill inthe art will appreciate that the thermodynamic lower limit of theliposome diameter will depend on a number of conditions, including pH,ionic strength, temperature, and type of lipid, among other factors. Insome instances, liposomes comprising cholesterol in amount of at least30 mole percent have an average diameter of about 45 nm. In someinstances, the liposomes are small unilamellar vesicles.

It is desirable to produce liposomes having the same molar ratio ofcomponent lipids as the lipid suspension from which the liposomes areformed. In other words, it is undesirable for the liposome productionmethod to result in a disproportionate loss of any component lipid. Asevidenced below in FIG. 52x12B, the methods of the invention in someembodiments produce liposomes wherein the lipid content of the lipidsuspension is substantially identical to the lipid content of theliposomes.

The liposome populations described herein have low polydispersities,generally having a polydispersity index that is less than 0.3, less than0.2, less than 0.15, or less than 0.10, as measured by DLS. In somecases, the liposomes comprising a sterol in amount of at least 30 molepercent have a polydispersity index of 0.20 or less.

Yield can be used to express the amount of lipids from the lipidsuspension that are not lost during filtration. In some embodiments, thelipid suspension is converted to liposomes with a yield of at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or at least about 90%.

III. SYSTEMS

Also disclosed are systems for preparing liposomes. Any of the liposomesdescribed in the Liposomes section above may be produced using thesystems disclosed herein. Further, any system component described in theMethods or Liposomes section above, such as housings, filters, syringes,syringe filters, orifices, etc. can be used in the systems provided.

In certain embodiments, a system for producing liposomes from a lipidsuspension comprises a filter assembly. In some embodiments, the filterassembly comprises two or more filters disposed in series with anorifice disposed between each pair of adjacent filters. In some cases,the filters are aligned such that their faces partially or completelyoverlap in the field of fluid flow. In some embodiments, the filters aresubstantially parallel. In some embodiments, the filters are aligned ina column.

In some instances, the system further comprises a filter housing havingan inlet and an outlet, wherein the filter assembly is disposed insidethe filter housing. In some instances, the filter assembly fits withinthe filter housing in such a manner that a fluid flowing through thefilter housing may not bypass the filters. In some instances, the filterassembly fits within the filter housing in such a manner that a fluidflowing through the filter housing must pass through the filter pores.

In some cases, the system further comprises a component to push, pull,or otherwise move a lipid suspension through the filter assembly. Thecomponent can be a plunger, a pump, a vacuum, a gravity feed, or anyother known component for moving a fluid through a system.

A flow rate is the volume of fluid that flows through the system perunit time. In some examples, the system has a flow rate of 1 mL/minuteor greater, 10 mL/minute or greater, 20 mL/minute or greater , 30mL/minute or greater, 50 mL/minute or greater, 100 m/min or greater, 1L/min or greater, 10 L/min or greater, 100 L/min or greater, or 1000L/min or greater.

The systems disclosed herein allow for the controlled production ofliposomes of decreasing diameter as a result of increasing the number offilters in series in the filter assembly, and also as a result ofincreasing the number of passes through the filter assembly. In somecases, the average diameter of the liposomes decreases with eachadditional pass through the filter assembly.

In some cases, each filter membrane has a thickness of 1 micron orgreater, 2 microns or greater, 3 microns or greater, 5 microns orgreater, 50 microns or greater, or 500 microns or greater.

In some embodiments, all filters in the filter assembly have the samediameter. In other embodiments, the filters have different diameters.The filters may be arranged according to increasing diameter, accordingto decreasing diameters, or randomly with respect to diameter.

Some previously-known systems for the production of liposomes requirethat the entire filter assembly be heated. In contrast, in someembodiments, the filters and filter housing are at ambient temperature.This ambient temperature can be room temperature (about 20-25° C.). Oneof skill in the art will appreciate that slight variations in thetemperature of the filters and/or filter housing may occur due to heattransfer from lipid suspension to the system. In general, the systemdoes not require a continuous supply of heat, or a supply of heatdirectly to the filter assembly.

The systems of the present invention are scalable. In some embodiments,the system has a static volume of 1-1000 microliters. In someembodiments, the system has a static volume of 1-1000 milliliters. Insome embodiments, the system has a static volume of 1-1000 liters.

A system 110 of the invention is shown in FIG. 1. Two filters 130 areconnected in series, with an orifice 140 disposed between. For thesystem depicted in FIG. 1, the diameter of orifice 140 is less than thediameter of filters 130. A lipid suspension 120 is filtered. A syringe150 is optionally used as the filter assembly inlet. Optionally, asyringe plunger may be used to move the lipid suspension 120 through thesystem.

Another system 210 of the invention is shown schematically in FIG. 2.Three filters 230 are connected in series, with an orifice 240 disposedbetween each two adjacent filters. For the system in FIG. 2, thediameter of orifices 240 is less than the diameter of filters 230. Thefilter housing 260 comprises an inlet 262 and an outlet 264. The lipidsuspension is moved through the filter housing by a component 270, whichcan be a pump. The lipid suspension may be recirculated through thesystem or may be removed from the system via valve 280.

IV. EXAMPLES Example 1. Comparison of Liposome Preparation via SerialFiltration Methods vs. Extrusion Methods

A 56:39:5 molar ratio of HSPC/Cholesterol/ DSPE-PEG5K lipid suspensionwas prepared by adding 12 mg of HSPC ((L-α-phosphatidylcholine,hydrogenated (Soy), Avanti Polar Lipids), 4 milligrams of cholesterol(Sigma-Aldrich), and 8 milligrams of DSPE-PEG5K(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethyleneglycol)-5000], ) to a 2:1 mixture of chloroform:methanol and dissolving.A thin film of these materials was generated by rotary evaporation at40° C. at 120 mbar. This film was desiccated overnight until completelydry. Hydration of the lipid film was conducted at 65° C. undersonication in 5 mL of 10 mM HEPES buffer (pH 7.4) for 1 h.

1 mL aliquots of the resulting lipid suspension were filtered throughfour syringe filters (100 nm pore size, 32 mm diameter, Supor filter,available from Pall Corporation) connected in series, or through a lipidextruder (available from Avanti Polar Lipids, Inc., Alabaster, Ala.)fitted with four stacked 100 nm membranes or four stacked 50 nmmembranes (available from Avanti Polar Lipids). In each case, thesuspension was filtered three times through the syringe filters orthrough the membranes. The diameter and polydispersity index of theresulting Example 1 liposomes are shown below in Table 1.

One pass through 4 filters with orifices disposed between the filtersresulting in liposomes having an average diameter of 60.49 nm and apolydispersity of 0.084 (Table 1, Example 1), whereas comparativeliposomes produced the filtering through four 100-nm membrane filtersstacked with no intervening orifices yielded liposomes with an averagediameter of 88.25 nm and a polydispersity of 0.150 (Table 1, ComparativeExample 1). Similarly, comparative liposomes produced the filteringthrough four 50-nm membrane filters stacked with no intervening orificesyielded liposomes with an average diameter of 82.03 nm and apolydispersity of 0.131 (Table 1, Comparative Example 2).

TABLE 1 Filter pore Liposome Method size (nm) size (nm) PDI Example 1Serial filtration 100 60.49 0.084 Comparative Example 1 Extrusion 10088.25 0.152 Comparative Example 2 Extrusion 50 82.03 0.131

Example 2. Serial Filtration with Increasing Number of Filters

Using the same lipid suspension as prepared in Example 1, 1 mL aliquotswere passed through 1, 2 or 4 syringe filters (100 nm pore size, 32 mmdiameter, Supor filter, available from Pall Corporation) connected inseries as described in Table 2. Smaller particle size is achieved by onepass through 4 filters in series (Table 2, Example 2.3, 70.62 nm) thanby four passes through an individual filter (Table 2, Example 2.1, 86.89nm), even though each lipid suspension passed through 4 filters.Further, a lower polydispersity index was achieved with filters inseries.

TABLE 2 Number of Number of Liposome filters in series passes size (nm)PDI Example 2.1 1 4 86.89 0.194 Example 2.2 2 2 83.39 0.181 Example 2.34 1 70.62 0.137FIG. 3 depicts average particle size and polydispersity of liposomesfiltered through 1, 2, or 4 filters in series as determined by DLS.

Example 3. Reproducible Preparation of Monodisperse Liposomes

FIG. 4 depicts reproducible preparation of ˜50 nm, ˜100 nm, and ˜200 nmliposomes, as determined by DLS.

Preparation of 200 nm Liposomes

A lipid film containing DPPE:Chol:DPPE-Cy5.5:DSPE-PEG5000 in a molarratio of 60:35:0.1:4.9 was prepared per the method of Example 1 washydrated at 69° C. without sonication for 1 h, and then was filteredthrough one syringe filter (450 nm pore size, 32 mm diameter, Suporfilter, available from Pall Corporation)) three times. The resulting thelipid solution was heated in the heated water bath for 5 minutes, andthen filtered through one syringe filter (200 nm pore size, 32 mmdiameter, Supor filter, available from Pall Corporation)) three times.

Preparation of 100 nm Liposomes

A lipid film containing DPPE:Chol:DPPE-Cy5.5:DSPE-PEG5000 in a molarratio of 60:35:0.1:4.9 was prepared per the method of Example 1 washydrated at 69° C. without sonication for 1 h, and then was filteredthrough one syringe filter (100 nm pore size, 32 mm diameter, Suporfilter, available from Pall Corporation) three times.

Preparation of 50 nm Liposomes

Syringe filter stacks were prepared by connecting the syringe filtershaving 32 mm diameter, available from Pall Corporation as in series asfollows:

TABLE 2 Identifier of Number of Pore Filter Filter Assembly filters inseries Size diameter Quantity “Stack 1” 3 200 nm 32 mm 2 “Stack 2” 3 100nm 32 mm 3

Each filter stack was pretreated by passing a PBS solution through thestack to pre-wet the membranes. The solution was then removed by passinga stream of air through the filter stack. A lipid solution containingDPPE:Chol:DPPE-Cy5.5:DSPE-PEG5000 in a molar ratio of 60:35:0.1:4.9 wasprepared per the method of Example 1, and was sonicated. While the lipidsolution was still hot (>63° C.), it was filtered through a Stack 1filter assembly followed by passing a stream of air through the stack.The resulting suspension was immediately passed through a second Stack 1filter assembly, followed by passing a stream of air through the stack.

The resulting suspension was sonicated with heat (69° C.) for 5 minutes,and then was filtered through a Stack 2 filter assembly followed bypassing a stream of air through the stack. The resulting suspension wassonicated with heat (69° C.) for 5 minutes, and then was filteredthrough the second Stack 2 filter assembly followed by passing a streamof air through the stack. The resulting suspension was sonicated withheat (69° C.) for 5 minutes, and then was filtered through the thirdStack 2 filter assembly followed by passing a stream of air through thestack.

Example 4. Linear Control of PEG Content

Lipid Suspension Preparation. A series of 12 mg:4 mg:0.1 mg:8 mgDPPE:Cholesterol: DPPE-Cy5.5:DSPE-PEG5000 lipid suspensions containing1.5, 2.5, and 5 mole percent PEG 5K, and a lipid suspension containing5% PEG 2000 were prepared.

1 mL aliquots of the resulting lipid suspensions were filtered asdescribed in Example 3. The PEG loading did not affect the particlesize, as shown in FIG. 5A. Linear control of PEG loading (at 1.5%, 2.5%,and 5%) was demonstrated by ¹H NMR, as shown in FIG. 5B. Further, thePEG lengths of 2000 and 5000 were incorporated at 5 mol % withoutaffecting particle size, as shown in FIG. 6. Determination by NMR wascarried out by integration of the peaks at 3.6 ppm and 0.6 ppm. Resultsshow a similar molar incorporation ratio of PEG relative to thecholesterol content for the given lipid content.

Example 5. Storage Stability

The liposomes of Example 4 demonstrated storage stability in PhosphateBuffer Saline (1×) at 4° C. for at least seven days, as shown in Table 3(PEG 2K=PEG2000, or 2000 g/mol; PEG 5K=PEG5000, or 5000 g/mol).

TABLE 4 Size on Size on Formulation Day 0 (nm), PDI Day 7 (nm), PDIExample 5.1 5% PEG 2K 63, 0.106 58, 0.119 Example 5.2 1.5% PEG 5K 76,0.105 78, 0.113 Example 5.3 2.5% PEG 5K 72, 0.165 70, 0.159 Example 5.45% PEG 5K 77, 0.113 74, 0.119

Example 6. Benefit of Additional Filters in Series

An HSPE:Cholesterol:DPPE-Cy5.5:DSPE-PEG 5000 (12 mg:4 mg:0.1 mg:8 mg)lipid suspension was prepared. 1 mL aliquots were passed through 1, 2,or 4 syringe filters (100 nm, 32 mm diameter, Supor filter, availablefrom Pall Corporation) connected in series, for a predetermined numberof passes as described in Table 5. Liposome particle size andpolydispersity resulting from the filtrations describe in Table 5 areshown in FIG. 7, which demonstrates that progressively smaller liposomesmay be produced by increasing the number of filters in series. Further,liposomes produced by 1×4 filtration are smaller that liposomes producedby 2×2 filtration, even though the lipid suspension passed through fourfilters in each instance. Further still, repeat passes through fourfilters in series further reduces the size of the liposomes (see 1×4,2×4, and 3×4). In all cases, the lipid suspensions contained 12 mg ofHSPC and 4 mg of cholesterol.

TABLE 5 Liposome Filtration method size(nm), PDI Example 6.1 1 × 1Single pass through one filter 95, 0.19 Example 6.2 1 × 2 Single passthrough two filters 85, 0.17 in series Example 6.3 2 × 2 Two passesthrough two filters 84, 0.17 in series Example 6.4 1 × 4 Single passthrough four filters 78, 0.13 in series Example 6.5 2 × 4 Two passesthrough four filters 76, 0.17 in series Example 6.6 3 × 4 Three passesthrough four filters 70, 0.17 in series

Example 7. Control of Liposome Diameter with Number of Passes

The lipid suspension of Example 6 was prepared, and 1 mL aliquots werepassed through two syringe filters (100 nm, 32 mm diameter, Suporfilter, available from Pall Corporation) connected in series, for aneven number of passes from 2-20, as shown in Table 6. Excellent controlof liposome particle size was achieved, with diameter decreasing withadditional passes until a size plateau was observed at 20 passes, asshown in FIG. 8.

TABLE 6 Passes Diameter (nm), PDI Example 7.1 2 94.25, 0.18 Example 7.24 83.39, 0.16 Example 7.3 6 78.93, 0.15 Example 7.4 8 74.93, 0.16Example 7.5 10  69.1, 0.12 Example 7.6 12 60.67, 0.15 Example 7.7 1456.69, 0.19 Example 7.8 16 64.62, 0.17 Example 7.9 18 54.24, 0.17Example 7.10 20 55.45450.16

V. EXEMPLARY EMBODIMENTS

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

1. A method of producing liposomes, comprising the steps of:

-   -   providing a lipid suspension comprising one or more component        lipids;    -   heating the lipid suspension to a temperature which is above the        phase transition temperature of the component lipids; and    -   passing the heated lipid suspension through a filter assembly,        wherein the filter assembly comprises two or more filters        connected in series, wherein an orifice is disposed between        adjacent filters, thereby producing the liposomes.

2. The method of embodiment 1, wherein the diameter of each orificedisposed between adjacent filters is less than or equal to about 70% ofthe diameter of the filters adjacent to the orifice.

3. The method of embodiment 1 or embodiment 2, wherein the filterscomprise alumina-based membranes, polycarbonate-based membranes,polyether sulfone-based membranes, or a combination thereof.

4. The method of any one of the preceding embodiments, wherein theaverage diameter of the liposomes is less than about 100 nm.

5. The method of any one of the preceding embodiments, wherein the ratioof the average filter pore size to the average liposome diameter is 1.6or greater.

6. The method of embodiment 5, wherein the average pore size of thefilters is about 100 nm and the average diameter of the liposomes isabout 50 nm.

7. The method of any one of the preceding embodiments, wherein thepressure in the filter assembly is less than 100 psi.

8. The method of any one of the preceding embodiments, wherein theaverage filter pore size is about 100 nm.

9. The method of any one of the preceding embodiments, wherein thefilter assembly comprises two filters connected in series.

10. The method of any one of embodiments 1-8, wherein the filterassembly comprises three filters connected in series.

11. The method of any one of embodiments 1-8, wherein the filterassembly comprises four filters connected in series.

12. The method of any one of the preceding embodiments, wherein thefilters are syringe filters.

13. The method of any one of the preceding embodiments, wherein theliposomes are small unilamellar vesicles.

14. The method of any one of the preceding embodiments, wherein theliposomes have a polydispersity index of 0.20 or less.

15. The method of any one of the preceding embodiments, wherein thelipid suspension comprises an amphiphilic lipid, a sterol, and a(polyethylene glycol)-lipid.

16. The method of embodiment 15, wherein the amphiphilic lipid isselected from the group consisting of hydrogenated soyphosphatidylcholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphatidylcholine; DMPC),1,2-distearoyl-sn-glycero-3-phosphocholine(distearoylphosphatidylcholine; DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine(dipalmitoylphosphatidylcholine; DPPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC),1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine,1,2-dipamiltoleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dielaidoyl-sn-glycero-3-phosphocholine,1,2-dipetroselenoyl-sn-glycero-3-phosphocholine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(palmitoyloleoylphosphatidylcholine; POPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC),1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC).

17. The method of embodiment 15 or embodiment 16, wherein the sterol ischolesterol or a cholesterol derivative.

18. The method of any one of embodiments 15-17, wherein the(polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine.

19. The method of any one of embodiments 15-18, wherein the amphiphiliclipid is hydrogenated soy phosphatidylcholine, the sterol ischolesterol, and the (polyethylene glycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K.

20. The method of any one of the preceding embodiments, wherein theliposomes have polyethylene glycol brush densities of >100,000polyethylene glycol chains per liposome having an average diameter of100 nm.

21. The method of any one of the preceding embodiments, wherein theliposomes comprise cholesterol in amount of at least 30 mole percent.

22. The method of any one of the preceding embodiments, wherein theaverage diameter of the liposomes is less than about 75 nm.

23. The method of any one of the preceding embodiments, wherein thelipid content of the lipid suspension is substantially identical to thelipid content of the liposomes.

24. The method of any one of the preceding embodiments, wherein thelipid suspension is passed through the filter assembly more than once.

25. The method of any one of the preceding embodiments, wherein theaverage diameter of the liposomes decreases with each additional passthrough the filter assembly.

26. The method of any one of the preceding embodiments, wherein thelipid suspension is converted to liposomes with a yield of at leastabout 70% (w/w).

27. A system for producing liposomes from a lipid suspension, comprisinga filter assembly comprising two or more filters disposed in series andan orifice disposed between each pair of adjacent filters.

28. The system of embodiment 27, further comprising a filter housinghaving an inlet and an outlet, wherein the filter assembly is disposedinside the filter housing.

29. The system of embodiment 27 or 28, further comprising a component tomove a lipid suspension through the filter assembly.

30. The system of any one of embodiments 27-31, wherein the system has aflow rate of 30 mL/minute or greater.

31. The system of any one of embodiments 27-30, wherein the filterscomprise alumina-based membranes, polycarbonate-based membranes,polyether sulfone-based membranes, or a combination thereof.

32. The system of any one of embodiments 27-31, wherein the filters havea thickness of 2 microns or greater.

33. The system of any one of embodiments 27-32, wherein the filters havethe same diameter.

34. The system of any one of embodiments 27-33, wherein the diameter ofeach orifice disposed between adjacent filters is less than or equal toabout 70% of the diameter of the filters adjacent to the orifice.

35. The system of any one of embodiments 27-34, wherein the filters andfilter housing are at ambient temperature during operation of thesystem.

36. The system of any one of embodiments 27-35, wherein the system has astatic volume of 1-1000 microliters.

37. The system of any one of embodiments 27-36, wherein the system has astatic volume of 1-1000 milliliters.

38. The system of any one of embodiments 27-36, wherein the system has astatic volume of 1-1000 liters.

39. Liposomes prepared according to the method of any one of embodiments1-26.

40. Liposomes prepared by a passing a lipid suspension through thesystem of any one of embodiments 27-38.

41. The liposomes of embodiment 39 or 40, wherein the ratio of theaverage filter pore size to the average diameter of the liposomes is 1.6or greater.

42. The liposomes of any one of embodiments 39-41, wherein the averagefilter pore size is about 100 nm and the average diameter of theliposomes is about 50 nm.

43. The liposomes of any one of embodiments 39-42, wherein the liposomeshave an average diameter of 100 nanometers or less.

45. A population of liposomes comprising a sterol in amount of at least30 mole percent, wherein the average diameter of the liposomes is lessthan 100 nm.

46. The liposomes of embodiment 44, wherein the average diameter of theliposomes is less than 75 nm.

47. The liposomes of embodiment 44, wherein the average diameter of theliposomes is less than 55 nm.

48. The liposomes of any one of embodiments 44-47, wherein the liposomesare small unilamellar vesicles.

49. The liposomes of any one of embodiments 44-48, wherein the liposomesfurther comprise an amphiphilic lipid, a (polyethylene glycol)-lipid, ora combination thereof.

50. The liposomes of any one of embodiments 44-49, wherein the sterol ischolesterol or a cholesterol derivative.

51. The liposomes of embodiment 49 or embodiment 50, wherein the(polyethylene glycol)-lipid is a (polyethyleneglycol)-phosphatidylethanolamine.

52. The liposomes of any one of embodiments 49-51, wherein theamphiphilic lipid is hydrogenated soy phosphatidylcholine, the sterol ischolesterol, and the (polyethylene glycol)-phospholipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-5K.

53. The liposomes of any one of embodiments 49-52, wherein the liposomeshave polyethylene glycol brush densities of >100,000 polyethylene glycolchains per liposome having an average diameter of 100 nm.

54. The liposomes of any one of embodiments 44-53, wherein the liposomeshave a polydispersity index of 0.20 or less.

55. The liposomes of embodiment 44, prepared according to method of anyone of embodiments 1-26.

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

1. A method of producing liposomes, comprising the steps of: providing alipid suspension comprising one or more component lipids; heating thelipid suspension to a temperature which is above the phase transitiontemperature of the component lipids; and passing the heated lipidsuspension through a filter assembly, wherein the filter assemblycomprises two or more filters connected in series, wherein an orifice isdisposed between adjacent filters, thereby producing the liposomes. 2.The method of claim 1, wherein the diameter of each orifice disposedbetween adjacent filters is less than or equal to about 70% of thediameter of the filters adjacent to the orifice.
 3. The method of claim1, wherein the filters comprise alumina-based membranes,polycarbonate-based membranes, polyether sulfone-based membranes, or acombination thereof.
 4. The method of claim 1, wherein the liposomeshave one or both of: (i) an average diameter less than about 100 nm orless than about 75 nm; and (ii) a polydispersity index of 0.20 or less.5-6. (canceled)
 7. The method of claim 1, wherein the pressure in thefilter assembly is less than 100 psi.
 8. The method of claim 1, whereinthe average filter pore size is about 100 nm.
 9. The method of claim 1,wherein the filter assembly comprises two, three, or four filtersconnected in series. 10-14. (canceled)
 15. The method of claim 1,wherein the lipid suspension comprises an amphiphilic lipid, a sterol,and a (polyethylene glycol)-lipid. 16-20. (canceled)
 21. The method ofclaim 1, wherein the liposomes comprise cholesterol in amount of atleast 30 mole percent.
 22. (canceled)
 23. The method of claim 1, whereinthe lipid content of the lipid suspension is substantially identical tothe lipid content of the liposomes. 24-26. (canceled)
 27. A system forproducing liposomes from a lipid suspension, comprising a filterassembly comprising two or more filters disposed in series and anorifice disposed between each pair of adjacent filters.
 28. The systemof claim 27, further comprising one or both of: (a) a filter housinghaving an inlet and an outlet, wherein the filter assembly is disposedinside the filter housing; and (b) a component to move a lipidsuspension through the filter assembly. 29-30. (canceled)
 31. The systemof claim 27, wherein the filters comprise alumina-based membranes,polycarbonate-based membranes, polyether sulfone-based membranes, or acombination thereof. 32-33. (canceled)
 34. The system of claim 27,wherein the diameter of each orifice disposed between adjacent filtersis less than or equal to about 70% of the diameter of the filtersadjacent to the orifice. 35-38. (canceled)
 39. Liposomes preparedaccording to the method of claim
 1. 40. Liposomes prepared by a passinga lipid suspension through the system of claim
 27. 41-43. (canceled) 44.A population of liposomes comprising a sterol in amount of at least 30mole percent, wherein the average diameter of the liposomes is less than100 nm.
 45. The liposomes of claim 44, wherein the liposomes have one orboth of: (i) an average diameter less than 75 nm or less than 55 nm; and(ii) a polydispersity index of 0.20 or less. 46-47. (canceled)
 48. Theliposomes of claim 44, wherein the sterol is cholesterol or acholesterol derivative.
 49. The liposomes of claim 44, wherein theliposomes further comprise an amphiphilic lipid, a (polyethyleneglycol)-lipid, or a combination thereof. 50-54. (canceled)